1. Technical Field
The present invention relates to a polypeptide having amidase activity, a gene thereof, a microorganism or a transformant that is able to produce the above polypeptide thereof, and a method for producing amidase using the above microorganism or the transformant thereof.
2. Background Art
Amidase (enzyme No. [EC3.5.1.4]) is an enzyme, which hydrolyzes a carboxylic amide to carboxylic acid and ammonia. It has been known that this enzyme has industrially useful activities. For example, this enzyme stereoselectively hydrolyzes a racemic α-amino acid amide to generate an optically active α-amino acid. Such an optically active α-amino acid is a compound that is useful as a synthetic intermediate of pharmaceuticals or a sweetener. It has also been reported that amidase catalyzes hydrolysis of an ester as a substrate (Non-Patent Document 1).
A large number of methods for producing optically active α-amino acids have been known. However, the mass production of D-α-amino acids via a fermentation method has been considered particularly difficult. Thus, it has been desired that an inexpensive efficient synthetic method be developed. For example, as a method for synthesizing D-α-amino acid via biological means, the following synthetic methods, which involve the use of a microorganism or an enzyme having amidase activity and optical resolution of a racemic α-amino acid amide, have been known.
(1) A method for producing a D-α-amino acid from a racemic α-amino acid amide, using activity for specifically hydrolyzing the D-α-amino acid amide of a microorganism belonging to genus Rhodococcus (Patent Document 1).
(2) A method for producing a D-α-amino acid from a racemic α-amino acid amide, using aminopeptidase produced by a microorganism belonging to genus Achromobacter, Corynebacterium, Flavobacterium, Bacillus, Micrococcus, Cellulomonas, Pseudomonas, Protaminobacter, Mycobacterium, Arthrobacter, or Streptomyces (Patent Document 2).(3) A method for producing D-alanine from a racemic alanine amide, using amidase produced by a microorganism belonging to genus Arthrobacter (Patent Documents 3 and 4).(4) A method for producing a D-α-amino acid from a racemic α-amino acid amide, using activity for specifically hydrolyzing the D-α-amino acid amide of a microorganism belonging to genus Achromobacter (Patent Document 5).(5) A method for producing a D-α-amino acid from a racemic α-amino acid amide, using a transformant having a D-amino acid amidase gene derived from a microorganism, which has been modified by genetic engineering (Patent Document 6).
However, in the case of the methods described in (1) to (4) above, since the hydrolytic activity of the microorganisms used is low, it cannot be said that these methods are satisfactory for industrial applications.
In addition, in the case of the method described in (5) above, only D-amino acid amidase derived from bacteria belonging to genus Ochrobactrum is described, and bacteria belonging to genus Arthrobacter are not described at all.
It has been generally known that bacteria belonging to genus Arthrobacter produce amidase. The enzyme has been purified and isolated, and the properties thereof have been then clarified (Patent Documents 3 and 4 and Non-Patent Documents 2 and 3). However, the amino acid sequence of the above enzyme and the nucleotide sequence of a gene encoding the above enzyme have not yet been reported.
[Patent Document 1] Japanese Patent Laid-Open No. 63-87998
[Patent Document 2] Japanese Patent Publication No. 7-106149
[Patent Document 3] U.S. Pat. No. 5,130,240
[Patent Document 4] U.S. Pat. No. 5,252,470
[Patent Document 5] Japanese Patent Laid-Open No. 2-234678
[Patent Document 6] Japanese Patent Laid-Open No. 2002-253256
[Non-Patent Document 1] Eur. J. Biochem., 2000, Vol. 267, p. 2028
[Non-Patent Document 2] Bioscience, Biotechnology and Biochemistry, 1992, Vol. 56, No. 12, p. 1980
[Non-Patent Document 3] Agricultural and Biological Chemistry, 1982, Vol. 46, No. 5, p. 1175